Biomedical subjects

C P Dietrich

Publications and source records attributed to C P Dietrich.

At least 19 recordsLinked to original sources

PubMed2006

EJ-ras oncogene transfection of endothelial cells upregulates the expression of syndecan-4 and downregulates heparan sulfate sulfotransferases and epimerase.

The EC rabbit endothelial cell line was transfected with the EJ-ras oncogene (EJ-ras EC). EJ-ras EC cells display over expression of the Ras oncogene, morphological changes and deregulation of the cell cycle, becoming more densely populated and serum-independent. In addition, EJ-ras-transfectant cells show higher levels of the syndecan-4 mRNA. In addition to the increase in the core protein, a parallel increase in the glycosylation of the syndecan-4 protein, a proteoglycan that bears heparan sulfate chains, also occurs. This increase is observed both for the heparan sulfate proteoglycan synthesized by the cells and for that secreted to the culture medium. This enhancement in heparan sulfate synthesis was observed through metabolic labeling of the cells, immunoprecipitation of syndecan-4 and heparitinases treatment. Furthermore, the EJ-ras-transfectant cells do not exhibit decreased synthesis of heparan sulfate during the G(1)-S phase transition, as observed for the parental cell line. Also, heparan sulfate synthesis is not stimulated by PMA as displayed by parental endothelial cells. Significant structural changes of heparan sulfate, such as decreased O-sulfation, were observed in the EJ-ras-transfected cells. Decreases in the mRNA levels of some enzymes (glucuronosyl C-5 epimerase, iduronosyl-2-O-sulfotransferase, glucosaminyl-6-O-sulfotransferase-1 and N-deacetylase/N-sulfotransferase-1), involved in the biosynthetic pathway of heparan sulfate, were also observed. The results suggest that overexpression of the EJ-ras oncogene alters the cell cycle, through signal transduction cascades, upregulates the expression of syndecan-4, and downregulates enzymes involved in the heparan sulfate biosynthesis related to chain modification, leading to the structural changes of the heparan sulfate syndecan-4 proteoglycan in endothelial cells.

Animals↗

PubMed2006

Specific structural features of syndecans and heparan sulfate chains are needed for cell signaling.

The syndecans, heparan sulfate proteoglycans, are abundant molecules associated with the cell surface and extracellular matrix and consist of a protein core to which heparan sulfate chains are covalently attached. Each of the syndecan core proteins has a short cytoplasmic domain that binds cytosolic regulatory factors. The syndecans also contain highly conserved transmembrane domains and extracellular domains for which important activities are becoming known. These protein domains locate the syndecan on cell surface sites during development and tumor formation where they interact with other receptors to regulate signaling and cytoskeletal organization. The functions of cell surface heparan sulfate proteoglycan have been centered on the role of heparan sulfate chains, located on the outer side of the cell surface, in the binding of a wide array of ligands, including extracellular matrix proteins and soluble growth factors. More recently, the core proteins of the syndecan family transmembrane proteoglycans have also been shown to be involved in cell signaling through interaction with integrins and tyrosine kinase receptors.

Animals↗

PubMed2003

Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors.

Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 micro/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 microM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 microM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 microM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).

Animals↗

PubMed2003

Enzyme and integrin expression by high and low metastatic melanoma cell lines.

Dissemination of a malignant tumour is the result of a cascade of events beginning with detachment of cells from primary tumour followed by extravasation and growth at secondary sites. The differences in metastatic ability could be attributed to properties intrinsic to the various tumour types. Thus the clonal selection of tumour cells from successive metastases apparently results in cells better equipped for survival and formation of colonies in secondary sites, indicating that survival is not a random phenomenon. Many studies of malignant cells have correlated the overexpression of adhesion receptors such as integrins and the production of cysteine proteases and glycosidases with the progression of malignancy. The interaction of cysteine proteases with basement membrane components has been implicated in tumour invasion, activation of hormones and growth factors. On the other hand, the expression of the heparanase gene and its protein has been associated with the metastatic potential of several human and mouse tumour cell lines. This study aimed to investigate the correlations between the metastatic properties of clones with high and low metastatic potential and their ability to adhere to the extracellular matrix and to degrade proteins and sulphated glycosaminoglycans present there. Clonal selection of the B16F10 cell line was performed, and the clones were examined for the expression of an integrin-type laminin receptor. A significantly higher level was detected in a high metastatic clone. Enzymatic assays showed higher activity for alpha-d-N-acetylglucosaminidase, beta-d-N-acetylgalactosaminidase and beta-d-glucuronidase in conditioned medium from low metastatic clones compared with that from high metastatic clones. However, higher endopeptidase activity was observed in conditioned medium from high metastatic clones. In summary, these results showed a positive correlation between high metastatic potential and endopeptidase secretion. Similarly, a positive correlation was observed between low metastatic cells and the secretion of glycosaminoglycan-degrading glycosidases.

Acetylglucosaminidase↗

PubMed2001

Heparin and a cyclic octaphenol-octasulfonic acid (GL-522-Y-1) bind with high affinity to a 47-kda protein from vascular endothelial cell surface and stimulate the synthesis and structural changes of heparan sulfate proteoglycan.

The effect of a cyclic octaphenol-octasulfonic acid (GL-522-Y-1), upon the synthesis of a heparan sulfate proteoglycan synthesized by endothelial cells (rabbit aorta and human umbilical vein) were studied. The cells were exposed to the compounds at various concentrations for different periods of time and the synthesized heparan sulfates analyzed by a combination of agarose gel electrophoresis and enzymatic degradation. The GL-522-Y-1, like heparin, change the sulfation pattern and stimulate two- to three-fold the synthesis of heparan sulfate proteoglycan secreted by rabbit and human endothelial cells in culture. GL-522-Y-1, besides being 100 times more active than heparin, also produces a significant enhancement of cell surface heparan sulfate in human vein endothelial cells. The effect of GL-522-Y-1 is completely abolished by methylation or acetylation of its free hydroxyl groups. Both heparin and GL-522-Y-1 have high affinity for a 47-kDa protein present at the surface of endothelial cells. These and other results lead us to speculate that the antithrombotic activity of heparin and GL522 "in vivo" could be related, at least in part, to the increased production of the heparan sulfate proteoglycan by endothelial cells.

Animals↗

PubMed2001

In vivo and in vitro cytotoxicity of brown spider venom for blood vessel endothelial cells.

The effect of brown spider (Loxosceles intermedia) venom on endothelial cells was investigated in vivo and in vitro. Morphological and ultrastructural observations by light microscopy and transmission electron microscopy showed that the venom acts in vivo upon vessel endothelial cells of rabbits that were intradermally injected, evoking vessel instability, cytoplasmic endothelial cell vacuolization, and blebs. Likewise, treatment of rabbit endothelial cells in culture with the venom led to loss of adhesion of the cells to the substrate. Endothelial cells in culture were metabolically radiolabeled with sodium [35S]-sulfate and the sulfated compounds (proteoglycans and sulfated proteins) from medium, cell surface, and extracellular matrix (ECM) were analyzed. Agarose gel electrophoresis and SDS-PAGE showed that the venom is active on the ECM and on cell surface proteoglycans, shedding these molecules into the culture medium. In addition, when purified heparan sulfate proteoglycan (HSPG) and purified laminin-entactin (LN/ET) complex were incubated with the venom we observed a partial degradation of the protein core of HSPG as well as the hydrolysis of entactin. The above results suggest that the L. intermedia venom has a deleterious effect on the endothelium of vessels both in vivo and in culture, removing important constituents such as HSPG and entactin that are involved in the adhesion of endothelial cells and of subendothelial ECM organization.

Animals↗

PubMed2001

Anticoagulant and antiprotease effects of a novel heparinlike compound from shrimp (Penaeus brasiliensis) and its neutralization by heparinase I.

Heparin is usually obtained from mammalian organs, such as beef lung, beef mucosa, porcine mucosa, and sheep intestinal mucosa. Because of the increased use of heparin in the production of low-molecular-weight heparin (LMWH), there is a growing shortage of the raw material needed to produce LMWHs. A previous report described the structural features of a novel LMWH from the shrimp (Penaeus brasiliensis). In order to compare anticoagulant and antiprotease effects of this heparin, global anticoagulant tests, such as the prothrombin time, activated partial thromboplastin time, thrombin time, and Heptest, were used. Amidolytic anti-Xa and anti-IIa activities were also measured. The relative susceptibility of this heparin to flavobacterial heparinase was also evaluated. The United States Pharmacopeia (USP) potency of shrimp heparin (SH) was found to be 28 U/mg. SH produced a concentration-dependent prolongation of all of the clotting tests and exhibited marked inhibition of FXa and FIIa. Heparinase treatment resulted in a marked decrease of the anticoagulant effects and neutralized the in vitro anti-IIa actions. However, the anti-Xa activities were only partially neutralized. Protamine sulfate was only partially effective in neutralizing the anticoagulant and antithrombin effects of SH. SH also produced marked prolongation of activated clotting time, which was neutralized by heparinase but not by protamine sulfate. These results suggest that SH is a strong anticoagulant with comparable properties to mammalian heparins and can be used in the development of clinically useful antithrombotic-anticoagulant drugs.

Animals↗

PubMed2001

A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins.

Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). The effect of this polymer on adhesion properties with specific extracellular matrix components was studied using several matrix proteins as substrates for cell attachment. Treatment with the polymer inhibited the adhesion of fibronectin to both CHO-K1 (2 x 10(5)) and CHO-745 (2 x 10(5) and 5 x 10(5)) cells. No effect was detected with laminin, using the two cell types. On the other hand, adhesion to vitronectin was inhibited in CHO-K1 cells and adhesion to type I collagen was inhibited in CHO-745 cells. In spite of this inhibition, the fucan did not affect either cell proliferation or cell cycle. These results demonstrate that this polymer is a new anti-adhesive compound with potential pharmacological applications.

Animals↗

PubMed2001

Development of new heparin-like compounds and other antithrombotic drugs and their interaction with vascular endothelial cells.

The anticlotting and antithrombotic activities of heparin, heparan sulfate, low molecular weight heparins, heparin and heparin-like compounds from various sources used in clinical practice or under development are briefly reviewed. Heparin isolated from shrimp mimics the pharmacological activities of low molecular weight heparins. A heparan sulfate from Artemia franciscana and a dermatan sulfate from tuna fish show a potent heparin cofactor II activity. A heparan sulfate derived from bovine pancreas has a potent antithrombotic activity in an arterial and venous thrombosis model with a negligible activity upon the serine proteases of the coagulation cascade. It is suggested that the antithrombotic activity of heparin and other antithrombotic agents is due at least in part to their action on endothelial cells stimulating the synthesis of an antithrombotic heparan sulfate.

Animals↗

PubMed2001

Heparan sulfate and control of cell division: adhesion and proliferation of mutant CHO-745 cells lacking xylosyl transferase.

We have examined the role of cell surface glycosaminoglycans in cell division: adhesion and proliferation of Chinese hamster ovary (CHO) cells. We used both wild-type (CHO-K1) cells and a mutant (CHO-745) which is deficient in the synthesis of proteoglycans due to lack of activity of xylosyl transferase. Using different amounts of wild-type and mutant cells, little adhesion was observed in the presence of laminin and type I collagen. However, when fibronectin or vitronectin was used as substrate, there was an enhancement in the adhesion of wild-type and mutant cells. Only CHO-K1 cells showed a time-dependent adhesion on type IV collagen. These results suggest that the two cell lines present different adhesive profiles. Several lines of experimental evidence suggest that heparan sulfate proteoglycans play a role in cell adhesion as positive modulators of cell proliferation and as key participants in the process of cell division. Proliferation and cell cycle assays clearly demonstrate that a decrease in the amount of glycosaminoglycans does not inhibit the proliferation of mutant CHO-745 cells when compared to the wild type CHO-K1, in agreement with the findings that both CHO-K1 and CHO-745 cells take 8 h to enter the S phase.

Animals↗

PubMed2000

Ultrastructural cytochemical characterization of collagen-associated proteoglycans in the endometrium of mice.

The decidual reaction in mice is characterized by the transformation of a specific population of endometrial fibroblasts into epithelioid cells, known as decidual cells. An important feature of decidualization in mice is a remarkable modification of the endometrial extracellular matrix. The present work is an ultrastructural cytochemical study of matrix with the purpose of analyzing the arrangement of collagen-associated proteoglycans (PGs) at various regions of nulliparous endometrium and of the antimesometrial decidua of mice using the cationic dye cuprolinic blue associated with enzymatic treatments with chondroitinase ABC, chondroitinase AC, and hyaluronidase. The staining with cuprolinic blue showed PGs as rods and granules of several sizes. Rods measuring 40-60 nm in length (named F2-rods) were apposed to thin collagen fibrils whereas granules were associated with thick collagen fibrils, particularly in the region occupied by mature decidual cells on the 7th day of pregnancy. The amount of granules was higher than that of F2-rods. Both F2-rods and granules were affected by chondroitinase ABC or AC treatment, indicating that they were PGs containing chondroitin sulfate and dermatan sulfate chains. However, the granules associated with thick collagen fibrils were more resistant to chondroitinase AC treatment than F2-rods, indicating the presence of dermatan sulfate chains that contain both L-iduronic and D-glucuronic acid sugar residues. We suggest that the differences of the nature and amount of PGs may be associated with the changes of the thickness of collagen fibrils observed during decidualization of the endometrium in the mouse.

Animals↗

PubMed2000

Distribution of sulfated glycosaminoglycans in the animal kingdom: widespread occurrence of heparin-like compounds in invertebrates.

Sulfated glycosaminoglycans were isolated from 23 species of 13 phyla of invertebrates and characterized by their electrophoretic migration in three different buffer systems coupled with enzymatic degradation using bacterial heparinase, heparitinases and chondroitinase AC. Heparan sulfate is a ubiquitous compound present in all species analyzed whereas chondroitin sulfate was present in 20 species and heparin-like compounds in 12 species of the invertebrates. The heparin-like compounds were purified from the echinoderm Mellita quinquisperforata (sand dollar) and the crustacean Ucides cordatus (crab) with anticoagulant activities of 60 and 52 IU/mg, respectively. Degradation of these heparins with heparinase produced significant amounts of the trisulfated disaccharide typical of mammalian heparins. This was confirmed by 13C-NMR spectroscopy of the crab heparin. An updated phylogenetic tree of the distribution of sulfated glycosaminoglycans in the animal kingdom is also presented.

Animals↗

PubMed2000

A novel heparan sulphate with high degree of N-sulphation and high heparin cofactor-II activity from the brine shrimp Artemia franciscana.

With the aid of heparinase and heparitinases from Flavobacterium heparinum and 13C and IH NMR spectroscopy it was shown that the heparan sulphate isolated from the brine shrimp Artemia franciscana exhibits structural features intermediate between those of mammalian heparins and heparan sulphates. These include an unusually high degree of N-sulphation (with corresponding very low degree of N-acetylation), a relatively high content of iduronic acid residues (both unsulphated and 2-O-sulphated) and a relatively low degree of 6-O-sulphation of the glucosamine residues. The major sequences (glucuronic acid-->N-sulphated glucosamine and glucuronic acid-->N, 6-disulphated glucosamine) are most probably arranged in blocks. Although exhibiting negligible anticlotting activity in the APTT and anti-factor Xa assays the A. franciscana heparan sulphate has a high heparin cofactor-II activity (about 1/3 that of heparin).

Animals↗

PubMed2000

Patients with head and neck tumors excrete a chondroitin sulfate with a low degree of sulfation: a new tool for diagnosis and follow-up of cancer therapy.

The chondroitin sulfate excreted in the urine of 10 patients with cancer of the head and neck and 27 healthy subjects was analyzed. The disaccharide products formed from chondroitin sulfate excreted by these 10 patients by action of chondroitinase ABC show a significant (P < 0.0001) relative increase of nonsulfated disaccharide (35.6% +/- 5.7%) when compared with the nonsulfated disaccharide (10.0% +/- 0.9%) present in the chondroitin sulfate of 27 healthy subjects. In 6 patients the structure of the excreted compound was analyzed up to 4 months after surgery. After removal of the cancer, the percent amounts of the nonsulfated disaccharide tend to approach the values found for the chondroitin sulfate of healthy subjects. A significant (P < 0.0001) change in the ratio of urinary chondroitin sulfate and heparan sulfate and a decrease in the electrophoretic migration of chondroitin sulfate were also observed. All of the patients with head and neck cancer analyzed so far have shown this structural anomaly of urinary chondroitin sulfate. This assay may be useful in the diagnosis and follow-up of cancer therapy.

Adult↗

PubMed2000

Effect of brown spider venom on basement membrane structures.

Loxoscelism or necrotic arachnidism are terms used to describe lesions and reactions induced by bites (envenomation) from spiders of the genus Loxosceles. Envenomation has been reported to provoke dermonecrosis and haemorrhage at the bite site and haemolysis, disseminated intravascular coagulation and renal failure. The purpose of this work was to study the effect of the venom of the brown spider Loxosceles intermedia on basement membrane structures and on its major constituent molecules. Light microscopy observations showed that L. intermedia venom obtained through electric shock, which reproduces two major signals of Loxoscelism in the laboratory, exhibits activity toward basement membrane structures in mouse Engelbreth-Holm-Swarm (EHS) sarcoma. Basement degradation was seen by a reduced periodic acid-Schiff (PAS) and alcian blue staining as well as by a reduced immunostaining for laminin when compared to control experiments. Electron microscopy studies confirmed the above results, showing the action of the venom on EHS-basement membranes and demonstrating that these tissue structures are susceptible to the venom. Using purified components of the basement membrane, we determined through SDS-PAGE and agarose gel that the venom is not active toward laminin or type IV collagen, but is capable of cleaving entactin and endothelial heparan sulphate proteoglycan. In addition, when EHS tissue was incubated with venom we detected a release of laminin into the supernatant, corroborating the occurrence of some basement membrane disruption. The venom-degrading effect on entactin was blocked by 1, 10-phenanthroline, but not by other protease inhibitors such as PMSF, NEM or pepstatin-A. By using light microscopy associated with PAS staining we were able to identify that 1,10-phenanthroline also inhibits EHS-basement membrane disruption evoked by venom, corroborating that a metalloprotease of venom is involved in these effects. Degradation of these extracellular matrix molecules and the observed susceptibility of the basement membrane could lead to loss of vessel and glomerular integrity, resulting in haemorrhage and renal problems after envenomation.

Animals↗

PubMed1999

Structural features and anticoagulant activities of a novel natural low molecular weight heparin from the shrimp Penaeus brasiliensis.

A natural low molecular weight heparin (8.5 kDa), with an anticoagulant activity of 95 IU/mg by the USP assay, was isolated from the shrimp Penaeus brasiliensis. The crustacean heparin was susceptible to both heparinase and heparitinase II from Flavobacterium heparinum forming tri- and di-sulfated disaccharides as the mammalian heparins. (13)C and (1)H NMR spectroscopy revealed that the shrimp heparin was enriched in both glucuronic and non-sulfated iduronic acid residues. The in vitro anticlotting activities in different steps of the coagulation cascade have shown that its anticoagulant action is mainly exerted through the inhibition of factor Xa and heparin cofactor II-mediated inhibition of thrombin. The shrimp heparin has also a potent in vivo antithrombotic activity comparable to the mammalian low molecular weight heparins.

Animals↗

PubMed1999

Oligosaccharide residues of Loxosceles intermedia (brown spider) venom proteins: dependence on glycosylation for dermonecrotic activity.

Loxosceles spp. (brown spider) envenomation has been reported to provoke dermonecrosis and haemorrhage at the bite site (a hallmark of accidents) and, to a lesser extent, thrombocytopenia, hemolysis and disseminated intravascular coagulation in some cases. Using lectin-immunolabeling, lectin-affinity chromatography, glycosidase and proteinase K treatments we were able to identify several venom N-glycosylated proteins with high-mannose oligosaccharide structures, complex-type glycoconjugates such as fucosylated glycans, but no galactose or sialic acid residues as complex sugars or glycosaminoglycan residues. Working with enzymatically or chemically deglycosylated venom we found that platelet aggregation (thrombocytopenic activity) as well as the fibronectinolytic and fibrinogenolytic (haemorrhagic) effects of the venom were sugar-independent when compared to glycosylated venom. Nevertheless, zymograph analysis in co-polymerized gelatin gels showed that enzymatic N-deglycosylation of loxolysin-B, a high-mannose 32-35 kDa glycoprotein of the venom with gelatinolytic metalloproteinase activity, caused a reduction of approximately 2 kDa in its molecular weight and a reduction of the gelatinolytic effect to a residual activity of 28% when compared to the glycosylated molecule, indicating a post-translational glycosylation-dependent gelatinolytic effect. Analysis of the dermonecrotic effect of the chemically or enzymatically N-deglycosylated venom detected only residual activity when compared with the glycosylated control. Thus, the present report suggests that oligosaccharide moieties play a role in the destructive effects of brown spider venom and opens the possibility for a carbohydrate-based therapy.

Animals↗

PubMed1999

New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli and 2-O-desulfated heparin.

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.

Bacterial Capsules↗